Association of liver injury from specific drugs, or groups of drugs, with polymorphisms in HLA and other genes in a genome-wide association study

: BACKGROUND AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; P = 2.4 × 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; P = 9.7 × 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; P = 4.8 × 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; P = 7.1 × 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. descent DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors. on behalf of International DILI consortium (iDILIC), Drug-induced liver injury network (DILIN) investigators and International Serious Adverse Events Consortium (iSAEC) Odd Ratio (OR); Roussel Uclaf Causality Assessment Method (RUCAM); Allele Frequency (AF); Hepatocellular (HC); Cholestatic-Mixed (CM); Expression quantitative trait loci (eQTL) Major Histocompatibility Complex (MHC); Minor allele frequency (MAF); Linkage Disequilibrium (LD); Human leukocyte antigen (HLA); Single Nucleotide Polymorphism (SNP) Abstract BACKGROUND & AIMS : We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS : We performed a GWAS of 862 persons with DILI and 10588 population-matched controls. The first set of cases was recruited prior to May 2009 in Europe (n=137) or the USA (n=274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the USA and South America. For the GWAS, we included only cases of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze human leukocyte antigen (HLA) genes and single nucleotide polymorphisms (SNPs). After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS : We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% CI, 1.9–3.8; P =2.4x10–8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6–2.5; P =9.7x10–9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant, genome wide, for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the LRBA gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6–2.7; CONCLUSIONS : In a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non–drug-specific risk factors.

European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze human leukocyte antigen (HLA) genes and single nucleotide polymorphisms (SNPs). After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. Previously, GWAS involving cohorts of DILI cases related to one particular drug only have resulted in identification of one or more drug-specific HLA risk alleles. 7-11 A large study involving 783 DILI cases due to a range of different drugs also resulted in a genome-wide significant HLA signal, but this association was abolished once 296 cases of DILI due to flucloxacillin and amoxicillin-clavulanate were excluded. 12 This partly reflects the fact that amoxicillin-clavulanate is a very common cause of DILI worldwide and flucloxacillin is an equally common cause in a number of Northern European countries. 13 Therefore, DNA collections from DILI cases generally will be highly enriched in cases relating to these two drugs, making detection of associations related to other compounds more difficult.

RESULTS
We have expanded our previous study of DILI caused by a range of different drugs, 12 and after excluding cases relating to amoxicillin-clavulanate and flucloxacillin, we have more than doubled the number of cases with additions from Europe, Australia, South America and the United States. We now report that HLA-A*33:01 is associated with risk of DILI, particularly due to terbinafine, fenofibrate and ticlopidine and especially with a cholestatic or mixed phenotype. We have also found novel nonmajor histocompatibility complex (MHC) related signals apparently shared across a range of different drugs; an intronic SNP, in the LPS-responsive vesicle trafficking, beach and anchor containing (LRBA) gene is associated with hepatocellular DILI and an intergenic SNP on chromosome 2, rs72631567, with DILI generally. An additional drug-specific genome-wide significant signal which could not be confirmed is also reported.

DILI discovery cohort
The cases in the study were from two separate recruitment phases. Europe and the USA were also considered) and where the DILI was not due to either flucloxacillin or amoxicillin-clavulanate were included (n=339). Clinical inclusion criteria for all cases were those described by Aithal et al. 15 Phase II case recruitment-DILIN. Details of the USA-based DILIN prospective study including IRB approval information have been described previously. 16 A total of 112 eligible new cases of European ancestry and ≥ 18 years were included in the current GWAS. These new cases were selected from the larger DILIN sample collection such that only cases relating to drugs also included among the iDILIC cases were represented. Laboratory inclusion criteria were as described previously. 16 Patients were excluded if there was known or suspected acetaminophen overdose, if there was a history of bone marrow or liver transplant prior to DILI onset or if there was a prior history of immune-related liver disease such as autoimmune hepatitis.

Statistical analysis
The effect of population structure was assessed through principal components analysis (PCA) using the smartPCA program from the EIGENSTRAT package (version 3.0). 22 Single marker and haplotype association analyses and heterogeneity test analyses were carried out by PLINK. 23 The statistical association of each marker, HLA alleles and SNPs, was determined in a logistic regression framework with scores for the first seven principal components as covariates under an additive model using PLINK. We used the same statistical test for sub-population analyses, using two, seven and ten most significant principal components as covariates in Italian, Spanish and North European populations, respectively. We set the genome-wide traditional significance p-value threshold to 5.0x10 -8 to correct for multiple testing. 24 When we obtained genome-wide significant signals, we tested for independent effects from the neighboring variants by including the most associated variants as a covariate and then testing the significance of others in the region. We also tested interaction effects among them by including interaction terms in the logistic regression. Differences in clinical characteristics among sample groups were tested by Fisher's exact test. All detailed analyses and Manhattan plots were performed with R (Version 3.0.2). 25 Regional plots were drawn by LocusZoom. 26

Clinical characteristics of the cases
Clinical details of the DILI cases included in the main GWAS are summarized in  Table S3.

Overall analysis
The discovery cohort included 862 European ancestry DILI cases (411 from phase I 12 and 451 from Phase II) and 10,588 controls. PCA showed that all cases (including those from South America) clustered within three major groups (Italian, Spanish and Northern European) and matched with the population controls ( Figure S1A).
Consistent with the previous study, 12 phase I cases were predominantly Northwest European. The most significant genome-wide associated SNPs were rs72631567 on chromosome 2 (OR=2.0, 95% CI =1.6-2.5, p-value=9.7x10 -9 ) and rs114577328 in the MHC region of chromosome 6 (OR=2.7, 95% CI=1.9-3.8, p-value=2.4x10 -8 )(See Figure 1A, Table 2 and Figures S2 and S3). Data for both SNPs had been obtained by imputation in cases and controls and subsequently validated by SNP typing (see Supplementary Methods). The associations were consistent among geographic clusters and study phases (Table S4) and not due to artefact/s of population structure, missing genotypes rate (Table S5)  For the chromosome 2 SNP rs72631567, breakdown by drug showed that 10 unrelated drug causes had an OR greater than 2.0 with at least two carriers (Table S6).
Including rs114577328 or A*33:01 as a covariate removed any association in the MHC region, indicating that there is only one MHC association signal ( Figure S5).
Breakdown by drug showed DILI due to terbinafine was most strongly associated with the HLA-A*33:01 signal (OR=40.5, 95% CI=12.5-131.4, p-value=6.7x10 -10 ) and a similarly strong association was seen with rs114577328 (OR=58.7, 95% CI=18.31-188.2, p-value=7.3x10 -12 , Figure 1B and Figure S6). As summarized in Table 3, in addition to terbinafine cases, cases due to six additional drugs showed an association with A*33:01 with p-values lower than 0.01. The largest case subset related to terbinafine but we found that A*33:01 was also a risk factor for ticlopidine

Analysis by type of injury and causative drugs
We further investigated the association of genotypes with particular patterns of DILI by grouping the cases into hepatocellular (HC) and cholestatic/mixed (CM) pattern.
The CM only terbinafine-specific OR increased two fold compared with the value for all terbinafine cases (OR=88.1, 95% CI = 19.3-402.4, p-value = 7.5x10 -9 ) since all the A*33:01 carriers belonged to this injury type. Following the injury correlation pattern established for terbinafine, A*33:01 appeared to be a stronger risk factor for CM injury than for HC injury also for fenofibrate, ticlopidine, enalapril and erythromycinrelated DILI. This was not the case for injury due to sertraline and methyldopa. These top seven drugs account for 51% (n=21) of all A*33:01 positive cases (Table S9).
Sixteen other drugs account for the rest of the carriers showing slight enrichment in CM phenotypes, which showed a marginal association with A*33:01 (OR = 2.6, 95% CI = 1.4-4.9, p-value = 0.003, Table S9 and Table S10).
We detected a new HC-specific genome-wide significant signal on chromosome 4 ( Figure 2B). The signal lies within the LRBA (LPS-responsive vesicle trafficking, beach and anchor containing) gene with the imputed variant rs28521457, located in an intronic region, the most significant SNP (OR=2.1, 95% CI=1.6-2.7, p-value=4.8x10 -9 )( Table 2 and Figure 2B). The allele frequency for this SNP in the CM cases (0.04) was comparable to that in controls with no evidence of association with this phenotype. The risk allele was carried by more than 4% of the HC cases in cases due to a total of 45 drugs but in general, there were no drug-specific signals (Table S11).
We also investigated associations with particular causative drugs or specific therapeutic classes where a group including more than 40 samples was available.
Detail on the groups studied is summarized in Table S12. Genome-wide significance was detected only for one group examined, the statins, with no significant signals for the other drug classes ( Figure S8 and Table S13). In the case of the statins, rs116561224, a common intergenic SNP on chromosome 18, was genome-wide significant (OR=5.4, 95% CI=3.0-9.5, p-value=7.1x10 -9 , Figure 2C and Figure S9) with the signal mainly driven by simvastatin (Table S14).

Confirmation of associations
The European cohort used to confirm the associations (n=283) had a wider range of causal drugs, mostly different from the discovery cohort (Table S1). Later in time, we had access to 15 additional cases relating specifically to the statin cohort.  (Table S16). This suggested a limited replication of the signal for these drugs.
Similarly, rs28521457 carriers seemed to be more common in the same subgroup of causal drugs in both cohorts (Table S17).
We also attempted to confirm the rs116561224 signal for statins. The number of additional cases available for this purpose was small (n=29, Table S1b) with only four simvastatin cases. None of the statin cases were positive for rs116561224 so the signal could not be confirmed.

Discussion
Our previous studies have been successful in identifying genetic risk factors for both flucloxacillin and amoxicillin-clavulanate DILI. 4,6 However, our most recent GWAS did not identify any risk factors that were common for DILI in general or specific genetic risk factors for DILI due to individual drugs which accounted for a smaller number of cases of DILI. 12 The current study included 451 additional cases of DILI due to a wide variety of causative drugs, including at least 10 DILI cases relating to each of 22 different drugs. This increase in numbers and the exclusion of the amoxicillin-clavulanate and flucloxacillin cases together with use of improved imputation methods has enabled the detection and confirmation of a novel genomewide significant signal relating to a relatively rare HLA class I allele A*33:01.
Though three other interesting signals were detected in the course of the study, an intergenic signal on chromosome 2, an intronic SNP in LRBA in HC cases only and a signal on chromosome 18 for statins, the failure to confirm these signals is a limitation. There are some indications that, as observed for HLA-A*33:01, the chromosome 2 and LRBA signals are shared across multiple unrelated drugs instead of being non-drug-specific risk variants. As supporting evidence, the chromosome 2 signal has been consistently associated in both replication and discovery cohorts with DILI due to ciprofloxacin, atorvastatin and mercaptopurine. There remains a possibility that replication could be achieved in a larger study involving a different mix of causative drugs but the degree of heterogeneity in drugs originally associated with the signals also increases the risk that these were chance observations.
Interestingly, unlike previously recognized HLA associations for DILI, A*33:01 also appears to be a risk factor for DILI due to several, structurally unrelated drugs. Our results also suggest that a haplotype comprising A*33:01, B*14:02, and C*08:02 may participate in concert to confer risk for DILI, as opposed to A*33:01 alone. However, because these alleles are so highly correlated, our current sample size does not allow us to distinguish between these possible explanations by genetic association evidence alone. This conceivable hypothesis could be further verified in a larger study, or by experiments with recombinant HLA proteins. 28 In the case of terbinafine where the A*33:01 association showed genome-wide significance for cases relating to this drug only, information on the underlying mechanism for hepatotoxicity is limited, N-dealkylation leads to the formation of an aldehyde metabolite, TBF-A, and this metabolite shows reactivity with glutathione. 29 It has been proposed that the GSH-adduct is transported across the canalicular membrane and concentrated in the bile where it may cause damage to biliary epithelial cells. There is limited data from the various case reports on an underlying inflammatory mechanism but it has been demonstrated that treatment of monocytes with terbinafine results in the release of the proinflammatory cytokines IL-8 and TNF-alpha. 30 Metabolism of terbinafine is complex involving several different cytochromes P450. 31 However, there was no evidence from the GWAS for a role for either CYP genes or innate immunity genes in the terbinafine DILI cases studied.
The other drugs showing the most convincing associations with A*33:01 were fenofibrate, ticlopidine and sertraline. Failure to see individual genome-wide significant associations with these drugs is likely to be due to fewer cases being available than for terbinafine. The A*33:01 association was seen for 3 of 7 cases due to fenofibrate, all with CM DILI. The literature on fenofibrate DILI is quite limited, but it appears that this drug is extensively metabolized, mainly by CYP3A4, 32 and there is a report of a drug interaction resulting in cholestatic injury 33 together with other isolated reports of idiosyncratic cholestatic DILI. 34 Ticlopidine-related DILI has been well studied previously, including two studies investigating genetic risk factors in Japanese individuals. Cholestatic liver injury also predominates in this form of DILI. 35,36 Ticlopidine is subject to extensive metabolism by several cytochrome P450 isoforms and carboxyesterase. 37 A study in rats suggests that adducts are formed following metabolism by cytochrome P450 with evidence for toxicity after biliary excretion of glutathione-conjugated metabolites via MRP2facilitated transport. 38 In previous studies, 22 Japanese patients with ticlopidine DILI showed an association with an HLA haplotype including A*33:03 (odds ratio 13). 39 In line with current observations, the association was strongest with cholestatic cases with 12 out of 14 cases positive for A*33:03. It should be noted that A*33:03 is relatively common in Japan with approximately 10 to 15% of individuals carrying this allele.
The observations on the HLA association for Japanese ticlopidine DILI cases were followed up by a report that those carrying a -2320T>C polymorphism in CYP2B6 were more susceptible to ticlopidine DILI due to high CYP2B6 expression (OR 2, p-value=0.04). 40 The CYP2B6 polymorphism (rs7254579) 41

is less frequent in
Europeans than in Asians (MAFcau = 0.29, MAFasian = 0.45) and its low effect size limited our ability to replicate the association in this small European ticlopidine DILI population, but the non-significant effect is in the same direction as the previous study (OR=2.8, 95% CI=0.7-10.18, p-value=0.11).
In contrast with the three drug examples above, sertraline is associated predominantly with HC DILI. [42][43][44] In line with this phenotypic association, there is evidence that sertraline can cause mitochondrial damage 28 and induce endoplasmic reticulum stress 45 in liver cells. There are parallels with a previous example of a HLA risk factor (DRB1*15:01) which is associated with predominantly CM DILI with amoxicillinclavulanate but HC DILI with lumiracoxib. 9,10 In line with the Japanese report of a role for A*33:03 in ticlopidine DILI 39  Most data for individual drug classes that were comparatively well represented in our cohort were entirely negative but the finding of a signal for statins which was driven by several class members was entirely novel. Similar to the more general signal seen on chromosome 2, the chromosome 18 is intergenic with the closest known gene, cadherin 19 located approx. 300000 bp downstream. Although functionally such a protein could be of relevance to the liver injury process, 59 any biological significance seems tenuous. The failure to confirm the signal in additional cases could be due to the availability of only a small cohort of additional cases which reflects the rarity of this form of DILI. 60 In conclusion, this study has detected a novel HLA association (HLA-A*33:01) in cases of DILI due to a number of different drugs, together with several novel non-HLA signals. Overall sensitivity and specificity of the A*33:01 allele as a predictor of DILI is low but our findings may be important for future drug treatment in cases of DILI due to one of the drugs for which the A*33:01 association is relevant. Follow-up studies are required to further explore the intergenic signal on chromosome 2, the biologically interesting signal in LRBA and the rs116561224 signal for statins in larger cohorts.

Figure 1
Manhattan plots displaying the association results of (A) the overall analysis (n=864); (B) terbinafine only cases (n=14 cases). SNPs in green have a significance level less than 5x10 -6 and red have a significance level less than 5x10 -8 .

Figure 2
Manhattan plot displaying the association results for (A) Cholestatic/Mixed only cases (n=323); (B) Hepatocellular only cases (n=474 cases); (C) Statin only cases (n=59). SNPs in green have a significance level less than 5x10 -6 and red have a significance level less than 5x10 -8 .